Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

• This invention relates to a process for the preparation of L-amino acids, in particular L-threonine, using strains of the Enterobacteriaceae family in which at least one or more of the genes chosen from the group consisting of dps, hns, lrp, pgm, fba, ptsG, ptsH, ptsl, err, mopB, ahpC and ahpF is (are) attenuated.
• L-Amino acids in particular L-threonine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
• Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms.
• Strains which are resistant to antimetabolites such as e.g. the threonine analogue ⁇ - amino- ⁇ -hydroxyvaleric acid (AHV) , or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as e.g. L-threonine, are obtained in this manner.
• Methods of the recombinant DNA technique have also been employed for some years for improving the strain of strains of the Enterobacteriaceae family which produce L- a ino acids, by amplifying individual amino acid biosynthesis genes and investigating the effect on the production.
• the object of the invention is to provide new measures for improved fermentative preparation of L-amino acids, in particular L-threonine.
• the invention provides a process for the preparation of L- amino acids, in particular L-threonine, using microorganisms of the Enterobacteriaceae family which in particular already produce L-amino acids and in which at least one or more of the nucleotide sequence (s) which code(s) for the genes dps, hns, lrp, pgm, fba, ptsG, ptsH, ptsl, err, mopB, ahpC and ahpF is (are) attenuated.
• L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L- asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L- isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L- histidine, L-lysine, L-tryptophan and L-arginine.
• L- Threonine is particularly preferred.
• the term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding enzyme (protein) or gene, and optionally combining these measures .
• the activity or concentration of the corresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the starting microorganism.
• the microorganisms which the present invention provides can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, optionally starch, optionally cellulose or from glycerol and ethanol. They are representatives of the Enterobacteriaceae family chosen from the genera Escherichia, Erwinia, Providencia and Serratia. The genera Escherichia and Serratia are preferred. Of the genus Escherichia the species Escherichia coli and of the genus Serratia the species Serratia marcescens are to be mentioned in particular. Suitable strains, which produce L-threonine in particular, of the genus Escherichia, in particular of the species Escherichia coli, are, for example
• Escherichia coli KCCM-10132 Escherichia coli KCCM-10132.
• Suitable L-threonine-producing strains of the genus Serratia in particular of the species Serratia marcescens, are, for example
• Strains from the Enterobacteriaceae family which produce L- threonine preferably have, inter alia, one or more genetic or phenotypic features chosen from the group consisting of: resistance to ⁇ -amino- ⁇ -hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to ⁇ - methylserine, resistance to diaminosuccinic acid, resistance to ⁇ -aminobutyric acid, resistance to borrelidin, resistance to rifampicin, resistance to valine analogues, such as, for example, valine hydroxamate, resistance to purine analogues, such as, for example, 6- dimethylaminopurine, a need for L-methionine, optionally a partial and compensable need for L-isoleucine, a need for meso-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptides, resistance to L-threonine, resistance to L
• microorganisms of the Enterobacteriaceae family produce L-amino acids, in particular L-threonine, in an improved manner after attenuation, in particular elimination, of at least one or more of the genes chosen from the group consisting of dps, hns, lrp, pgm, fba, ptsG, ptsH, ptsl, err, mopB, ahpC and ahpF.
• endogenous genes are in general preferred.
• endogenous genes or “endogenous nucleotide sequences” is understood to mean the genes or nucleotide sequences present in the population of a species .
• dps gene Description: Global regulator, hunger conditions, DNA binder protein
• DNA-binding protein HLP-II (HU, BH2, HD, NS) ; pleiotropic regulator (histone-like protein)
• lrp gene Description: Regulator for the leucine regulon and high- affinity transport systems of branched- chain amino acids (leucine-responsive regulatory protein)
• fba gene Description: Fructose bisphosphate aldolase (class II) EC No . : 4.1.2.13 Reference: Alefounder et al . , Biochemical Journal
• ptsG gene Description: PTS system, glucose-specific IIBC component
• ptsH gene Description: phosphohistidine protein hexose phosphotransferase, Phosphocarrier HPr protein of the phosphotransferase-Systems (PTS)
• ptsl gene Description: Phosphoenolpyruvat-Protein-
• Phosphotransferase-Systems EC No. : 2.7.3.9 Reference: Saffen et al.; Journal of Biological
• glucose-specific IIA component phospho- carrier protein for. glucose
• Phosphotransferase-Systems PPS
• mopB gene Description: chaperone GroES, binds to heat-shock protein Hsp60 in the presence of Mg-ATP, suppresses ATPase activity Reference: Chandrasekhar et al.; Journal of Biological Chemistry 261(26) : 12414-9 (1986) LaRossa and Van Dyk; Molecular Microbiology 5(3) : 529-534 (1991) Accession No. : AE000487 Alternative gene names: groE, groES, hdh, tabB
• ahpC gene Description: C22- subunit of the alkyl hydroperoxide reductase, detoxification of hydroperoxides
• ahpF gene Description: F52a-subunit of the alkyl hydroperoxide reductase; detoxification of hydroperoxides Reference: Ferrante et al . ; Proceedings of the National Academy of Sciences USA • 92(17) .7617-21 (1995) Poole und Ellis; Biochemistry 35(l):56-64 (1996) Nishiyama et al . ; Journal of Bacteriology 183(8) :2431-2438 (2001)
• nucleic acid sequences can be found in the databanks of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) , the nucleotide sequence databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany or Cambridge, UK) or the DNA databank of Japan (DDBJ, Mishima, Japan) .
• NCBI National Center for Biotechnology Information
• EMBL European Molecular Biologies Laboratories
• EMBL European Molecular Biologies Laboratories
• DDBJ Mishima, Japan
• expression of the genes or the catalytic properties of the enzyme proteins can be reduced or eliminated.
• the two measures can optionally be combined.
• the reduction in gene expression can take place by suitable culturing, by genetic modification (mutation) of the signal structures of gene expression or also by the antisense-RNA technique.
• Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators .
• Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, "missense mutations” or “nonsense mutations” are referred to. Insertions or deletions of at least one base pair in a gene lead to "frame shift mutations", which lead to incorrect amino acids being incorporated or translation being interrupted prematurely. If a stop codon is formed in the coding region as a consequence of the mutation, this also leads to a premature termination of the translation. Deletions of several codons typically lead to a complete loss of the enzyme activity. Instructions on generation of such mutations are prior art and can be found in known textbooks of genetics and molecular biology, such as e.g.
• Suitable mutations in the genes can be incorporated into suitable strains by gene or allele replacement.
• a conventional method is the method, described by Hamilton et al. (Journal of Bacteriology 171: 4617 - 4622 (1989)), of gene replacement with the aid of a conditionally replicating pSClOl derivative pMAK705.
• Other methods described in the prior art such as, for example, those of Martinez-Morales et al. (Journal of Bacteriology 181: 7143- 7148 (1999)) or those of Boyd et al . (Journal of Bacteriology 182: 842-847 (2000)), can likewise be used. It is also possible to transfer mutations in the particular genes or mutations which affect expression of the particular genes into various strains by conjugation or transduction.
• L- amino acids in particular L-threonine
• strains of the Enterobacteriaceae family to enhance one or more enzymes of the known threonine biosynthesis pathway or enzymes of anaplerotic metabolism or enzymes for the production of reduced nicotinamide adenine dinucleotide phosphate, in addition to the attenuation of one or more of the genes chosen from the group consisting of dps, hns, lrp, pgm, fba, ptsG, ptsH, ptsl, err, mopB, ahpC and ahpF.
• enhancement in this connection describes the increase in the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or a gene which codes for a corresponding enzyme or protein with a high activity, and optionally combining these measures .
• the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism.
• L- amino acids in particular L-threonine
• the genes chosen from the group consisting of dps, hns, lrp, pgm, fba, ptsG, ptsH, ptsl, err, mopB, ahpC and ahpF for one or more of the genes chosen from the group consisting of • the tdh gene which codes for threonine dehydrogenase (Ravnikar and Somerville, Journal of Bacteriology 169: 4716-4721 (1987)),
• L- amino acids in particular L-threonine
• dps hns
• lrp pgm
• fba ptsG
• ptsH ptsl
• err mopB
• mopB mopB
• ahpC ahpF
• microorganisms produced according to the invention can be cultured in the batch process (batch culture) , the fed batch (feed process) or the repeated fed batch process (repetitive feed process) .
• batch culture batch culture
• feed process fed batch
• repetitive feed process repetition feed process
• the culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C. , USA, 1981).
• Sugars and carbohydrates such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and optionally cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substance can be used individually or as a mixture.
• oils and fats such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat
• fatty acids such as e.g. palmitic acid, stearic acid and linoleic acid
• alcohols such as e.g. glycerol and ethanol
• organic acids such as e.g. acetic acid
• Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea
• inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen.
• the sources of nitrogen can be used individually or as a mixture.
• Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus.
• the culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth.
• essential growth substances such as amino acids and vitamins, can be employed in addition to the above-mentioned substances.
• Suitable precursors can moreover be added to the culture medium.
• the starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.
• Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture.
• Antifoams such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam.
• Suitable substances having a selective action e.g. antibiotics, can be added to the medium to maintain the stability of plasmids.
• oxygen or oxygen-containing gas mixtures such as e.g. air, are introduced into the culture.
• the temperature of the culture is usually 25 a C to 45 a C, and preferably 30 a C to 40 fi C.
• L-amino acids or L-threonine Culturing is continued until a maximum of L-amino acids or L-threonine has formed. This target is usually reached within 10 hours to 160 hours.
• the analysis of L-amino acids can be carried out by anion exchange chromatography with subsequent ninhydrin derivation, as described by Spackman et al. (Analytical Chemistry, 30: 1190-1206 (1958)), or it can take place by reversed phase HPLC as described by Lindroth et al . (Analytical Chemistry 51: 1167-1174 (1979)).
• the process according to the invention is used for the fermentative preparation of L-amino acids, such as, for example, L-threonine, L-isoleucine, L-valine, L-methionine, L-homoserine and L-lysine, in particular L-threonine.
• L-amino acids such as, for example, L-threonine, L-isoleucine, L-valine, L-methionine, L-homoserine and L-lysine, in particular L-threonine.

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• 2001
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